B-cell lymphoma/leukaemia 10 and angiotensin II-induced kidney injury.

AIMS: B-cell lymphoma/leukaemia 10 (Bcl10) is a member of the CARMA-Bcl10-MALT1 signalosome, linking angiotensin (Ang) II, and antigen-dependent immune-cell activation to nuclear factor kappa-B signalling. We showed earlier that Bcl10 plays a role in Ang II-induced cardiac fibrosis and remodelling, independent of blood pressure. We now investigated the role of Bcl10 in Ang II-induced renal damage. METHODS AND RESULTS: Bcl10 knockout mice (Bcl10 KO) and wild-type (WT) controls were given 1% NaCl in the drinking water and Ang II (1.44 mg/kg/day) for 14 days. Additionally, Bcl10 KO or WT kidneys were transplanted onto WT mice that were challenged by the same protocol for 7 days. Kidneys of Ang II-treated Bcl10 KO mice developed less fibrosis and showed fewer infiltrating cells. Nevertheless, neutrophil gelatinase-associated lipocalin (Ngal) and kidney injury molecule (Kim)1 expression was higher in the kidneys of Ang II-treated Bcl10 KO mice, indicating exacerbated tubular damage. Furthermore, albuminuria was significantly higher in Ang II-treated Bcl10 KO mice accompanied by reduced glomerular nephrin expression and podocyte number. Ang II-treated WT mice transplanted with Bcl10 KO kidney showed more albuminuria and renal Ngal, compared to WT- > WT kidney-transplanted mice, as well as lower podocyte number but similar fibrosis and cell infiltration. Interestingly, mice lacking Bcl10 in the kidney exhibited less Ang II-induced cardiac hypertrophy than controls. CONCLUSION: Bcl10 has multi-faceted actions in Ang II-induced renal damage. On the one hand, global Bcl10 deficiency ameliorates renal fibrosis and cell infiltration; on the other hand, lack of renal Bcl10 aggravates albuminuria and podocyte damage. These data suggest that Bcl10 maintains podocyte integrity and renal function.

Reduced response to chronic mild stress in PACAP mutant mice is associated with blunted FosB expression in limbic forebrain and brainstem centers.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in stress adaptation with potential relevance in mood disorder management. PACAP deficient (KO) mice on CD1 background were shown to have depression-like phenotype. Here we aimed at investigating effects of chronic variable mild stress (CVMS) in non-injected, vehicle and imipramine-treated KO mice vs. wildtype (WT) counterparts. We hypothesized reduced FosB neuronal activity in stress-related centers, altered activity and peptide/neurotransmitter content of corticotropin-releasing factor (CRF) cells of the oval (ovBST) bed nucleus of stria terminalis (BST), urocortin 1 (Ucn1) neurons of centrally projecting Edinger-Westphal nucleus (cpEW) and serotonin (5HT) cells of dorsal raphe (DR) in PACAP deficiency. CVMS caused decreased body weight and increased adrenal size, corticosterone (CORT) titers and depression-like behavior in WT mice, in contrast to KO animals. CVMS increased FosB in the central (CeA) and medial amygdala, dorsomedial (dmBST), ventral (vBST), ovBST, CA1 area, dentate gyrus (DG), ventral lateral septum, parvo- (pPVN) and magnocellular paraventricular nucleus, lateral periaqueductal gray, cpEW and DR. Lack of PACAP blunted the CVMS-induced FosB rise in the CeA, ovBST, dmBST, vBST, CA1 area, pPVN and DR. The CVMS-induced FosB expression in ovBST-CRF and cpEW-Ucn1 neurons was abolished in KO mice. Although CVMS did not induce FosB in 5HT-DR neurons, PACAP KO mice had increased 5HT cell counts and 5HT content. We conclude that PACAP deficiency affects neuronal reactivity in a brain area-specific manner in stress centers, as well as in ovBST-CRF, cpEW-Ucn1 and 5HT-DR neurons leading to reduced CVMS response and altered depression level.

Role of Cystathionine Gamma-Lyase in Immediate Renal Impairment and Inflammatory Response in Acute Ischemic Kidney Injury.

Hydrogen sulfide (H2S) is known to act protectively during renal ischemia/reperfusion injury (IRI). However, the role of the endogenous H2S in acute kidney injury (AKI) is largely unclear. Here, we analyzed the role of cystathionine gamma-lyase (CTH) in acute renal IRI using CTH-deficient (Cth(-/-)) mice whose renal H2S levels were approximately 50% of control (wild-type) mice. Although levels of serum creatinine and renal expression of AKI marker proteins were equivalent between Cth(-/-) and control mice, histological analysis revealed that IRI caused less renal tubular damage in Cth(-/-) mice. Flow cytometric analysis revealed that renal population of infiltrated granulocytes/macrophages was equivalent in these mice. However, renal expression levels of certain inflammatory cytokines/adhesion molecules believed to play a role in IRI were found to be lower after IRI only in Cth(-/-) mice. Our results indicate that the systemic CTH loss does not deteriorate but rather ameliorates the immediate AKI outcome probably due to reduced inflammatory responses in the kidney. The renal expression of CTH and other H2S-producing enzymes was markedly suppressed after IRI, which could be an integrated adaptive response for renal cell protection.

Role of mitochondrial network stabilisation by a human small heat shock protein in tumour malignancy.

Previously, we found that the unconventional small human heat-shock protein HSPB11 inhibits cell death by HSP90 mediated cholesterol-rich membrane microdomain dependent activation of phosphatidylinositol-3 kinase/protein kinase B pathway and by stabilising the mitochondrial membrane systems. Also, progressive cytoplasmic expression of HSPB11 correlated with brain tumor malignancy. In the present study we investigated how cytoplasmic abundance of HSPB11 augments tumor malignancy. We up- and downregulated the cytoplasmic level of HSPB11 before paclitaxel exposure in NIH3T3 and HeLa cells, which normally express low and high levels, respectively, of the HSPB11 protein. We examined the paclitaxel-mediated induction of cell death, mitochondrial fission, HSPB11 mitochondrial translocation and inhibitory phosphorylation of dynamin-like protein-1 (DLP1). We found that increasing cytoplasmic abundance of HSPB11 in NIH3T3 cells protected against paclitaxel-induced apoptosis, while suppressing HSPB11 in HeLa cells sensitised the cells toward paclitaxel. Also, paclitaxel enhanced mitochondrial translocation of HSPB11 in wild type HeLa but not in NIH3T3 cells. More importantly, increased cytoplasmic level of HSPB11 in NIH3T3 cells enhanced the inhibitory phosphorylation of DLP1 and attenuated paclitaxel-induced mitochondrial fission. All these results suggest that increased cytoplasmic abundance of HSPB11 augments inhibitory phosphorylation of DLP1 thereby reduces mitochondrial fission that eventually leads to decreased apoptosis. This novel mechanism may explain the resistance to apoptosis and increased malignancy of HSPB11-overexpressing tumours. The clinical significance of this mechanism has already been highlighted by the finding that the kinase inhibitor tyrphostin A9 induces cancer cell death by DLP1-mediated mitochondrial fragmentation.

Absence of Nkx2-3 homeodomain transcription factor reprograms the endothelial addressin preference for lymphocyte homing in Peyer's patches.

Although the homing of lymphocytes to GALT has been extensively studied, little is known about how high endothelial venules (HEVs) within Peyer's patches (PPs) are patterned to display dominantly mucosal addressin cell adhesion molecule 1 (MAdCAM-1). In this study, we report that Nkx2-3-deficient mice show gradual loss of MAdCAM-1 in PPs postnatally and increased levels of mRNA for peripheral lymph node addressin (PNAd) backbone proteins as well as enhanced expression of MECA79 sulfated glycoepitope at the luminal aspect of HEVs, thus replacing MAdCAM-1 with PNAd. Induction of PNAd in mutant PPs requires lymphotoxin ß receptor activity, and its upregulation needs the presence of mature T and B cells. Furthermore, treatment with MECA-79 anti-PNAd mAb in vivo effectively blocks lymphocyte homing to mutant PPs. Despite the replacement of MAdCAM-1 by PNAd in HEV endothelia, lymphocytes could efficiently home to PPs in mutant mice. We conclude that although Nkx2-3 activity controls the addressin balance of HEVs in GALT, the general HEV functionality is preserved independently from Nkx2-3, indicating a substantial plasticity in the specification of GALT HEV endothelium.

Bcl10 mediates angiotensin II-induced cardiac damage and electrical remodeling.

Angiotensin (Ang) II is a potent mediator of both hypertension and cardiac damage; however, the mechanisms by which this occur remain unclear. B-cell lymphoma/leukemia 10 (Bcl10) is a member of the CBM signalosome, which links Ang II and nuclear factor-κB signaling. We hypothesized that Bcl10 is pivotal in the pathogenesis of Ang II-induced cardiac damage. Ang II infusion in mice lacking Bcl10 resulted in reduced cardiac fibrosis, less cellular infiltration, and improved arrhythmogenic electric remodeling, despite a similar degree of hypertension or cardiac hypertrophy. Adoptive transfer of bone marrow (BM), whereby Bcl10 knockout or wildtype BM was transferred to their opposite genotype recipients, revealed the dual importance of Bcl10 within both cardiac and immune cells. Loss of Bcl10 in cardiac cells resulted in reduced expression of genes important for the adhesion and recruitment of immune cells. In vitro experiments demonstrated that adhesion of monocytes to Ang II-treated endothelial cells also required Bcl10. Additionally, Bcl10 deficiency in macrophages reduced their intrinsic migratory ability. To address the role of BM-derived fibroblasts in the formation of cardiac fibrosis, we explored whether Bcl10 is also important for the infiltration of BM-derived (myo)fibroblasts into the heart. The transfer of green fluorescent protein positive wildtype BM into Bcl10 knockout recipient mice revealed a reduced number of noncardiac (myo)fibroblasts compared with those wildtype recipients. Our results demonstrate the significant role of Bcl10 in multiple cell types important for the generation of Ang II-induced cardiac damage and electric remodeling and may provide a new avenue for therapeutic intervention.

Overexpression of CREB protein protects from tunicamycin-induced apoptosis in various rat cell types.

Endoplasmic reticulum (ER) stress plays an essential role in unfolded protein response induced apoptosis contributing to several pathological conditions. Glycogen synthase kinase-3ß (GSK-3ß) plays a central role in several apoptotic signaling, including ER stress, as the active form of GSK-3ß induces apoptosis. The phosphorylation of cAMP responsive element (CRE) binding protein (CREB) Ser-133 (S133) residue is the end-point of various signaling pathways, like growth factor signaling, while the Ser-129 (S129) residue is phosphorylated by GSK-3ß. The significance of the ubiquitously expressed transcription factor CREB is demonstrated in prolonged, tunicamycin (TM)-induced ER stress in this study. In the experiments wild-type (wt) CREB, S129Ala, S133Ala or S129Ala-S133Ala mutant CREB expressing PC12 rat pheochromocytoma cell lines showed increased survival under TM-evoked prolonged ER stress compared to wtPC12 cells. After TM treatment ER stress was activated in all PC12 cell types. Lithium and SB-216763, the selective, well-known inhibitors of GSK-3ß, decreased TM-induced apoptosis and promoted cell survival. The proapoptotic BH3-only Bcl-2 family member Bcl-2-interacting mediator of cell death (Bim) level was decreased in the different CREB overexpressing PC12 cells as a result of TM treatment. CREB overexpression also inhibited the sequestration of Bim protein from tubulin molecules, as it was demonstrated in wtPC12 cells. Transient expression of wtCREB diminished TM-induced apoptosis in wtPC12, Rat-1 and primary rat vascular smooth muscle cells. These findings demonstrate a novel role of CREB in different cell types as a potent protector against ER stress.

Gene expression profiling in PC12 cells infected with an oncolytic Newcastle disease virus strain.

Although the oncolytic potential of natural, non-engineered Newcastle disease virus (NDV) isolates are well-known, cellular mechanisms determining NDV sensitivity of tumor cells are poorly understood. The aim of the present study was to look for gene expression changes in PC12 pheochromocytoma cells infected with an attenuated NDV strain that may be related to NDV susceptibility. PC12 cells were infected with the NDV strain MTH-68/H for 12h at a titer corresponding to the IC50 value. Total cytoplasmic RNA samples isolated from control and MTH-68/H-infected cells were analyzed using a rat specific Affymetrix exon chip. Genes with at least 2-fold increase or decrease in their expression were identified. MTH-68/H-induced gene expression changes of 9 genes were validated using quantitative reverse transcriptase PCR. A total of 729 genes were up- and 612 genes were down-regulated in PC12 cells infected with MTH-68/H. Using the DAVID functional annotation clustering tool, the up- and down-regulated genes can be categorized into 176 and 146 overlapping functional gene clusters, respectively. Gene expression changes affecting the most important signaling mechanisms (Toll-like receptor signaling, RIG-I-like receptor signaling, interferon signaling, interferon effector pathways, apoptosis pathways, endoplasmic reticulum stress pathways, cell cycle regulation) are analyzed and discussed in detail in this paper. NDV-induced gene expression changes described in this paper affect several regulatory mechanisms and dozens of putative key proteins that may determine the NDV susceptibility of various tumors. Further characterization of these proteins may identify susceptibility markers to predict the chances of virotherapeutic treatment of human tumors.

A quinazoline-derivative compound with PARP inhibitory effect suppresses hypertension-induced vascular alterations in spontaneously hypertensive rats.

AIMS: Oxidative stress and neurohumoral factors play important role in the development of hypertension-induced vascular remodeling, likely by disregulating kinase cascades and transcription factors. Oxidative stress activates poly(ADP-ribose)-polymerase (PARP-1), which promotes inflammation and cell death. We assumed that inhibition of PARP-1 reduces the hypertension-induced adverse vascular changes. This hypothesis was tested in spontaneously hypertensive rats (SHR). METHODS AND RESULTS: Ten-week-old male SHRs and wild-type rats received or not 5mg/kg/day L-2286 (a water-soluble PARP-inhibitor) for 32 weeks, then morphological and functional parameters were determined in their aortas. L-2286 did not affect the blood pressure in any of the animal groups measured with tail-cuff method. Arterial stiffness index increased in untreated SHRs compared to untreated Wistar rats, which was attenuated by L-2286 treatment. Electron and light microscopy of aortas showed prominent collagen deposition, elevation of oxidative stress markers and increased PARP activity in SHR, which were attenuated by PARP-inhibition. L-2286 treatment decreased also the hypertension-activated mitochondrial cell death pathway, characterized by the nuclear translocation of AIF. Hypertension activated all three branches of MAP-kinases. L-2286 attenuated these changes by inducing the expression of MAPK phosphatase-1 and by activating the cytoprotective PI-3-kinase/Akt pathway. Hypertension activated nuclear factor-kappaB, which was prevented by PARP-inhibition via activating its nuclear export. CONCLUSION: PARP-inhibition has significant vasoprotective effects against hypertension-induced vascular remodeling. Therefore, PARP-1 can be a novel therapeutic drug target for preventing hypertension-induced vascular remodeling in a group of patients, in whom lowering the blood pressure to optimal range is harmful or causes intolerable side effects.

Partial rescue of geldanamycin-induced TrkA depletion by a proteasome inhibitor in PC12 cells.

In this work we tried to identify mechanisms that could explain how chemical inhibition of heat-shock protein 90 reduces nerve growth factor signaling in rat pheochromocytoma PC12 cells. Geldanamycin is an antibiotic originally discovered based on its ability to bind heat-shock protein 90. This interaction can lead to the disruption of heat-shock protein 90-containing multimolecular complexes. It can also induce the inhibition or even degradation of partner proteins dissociated from the 90 kDa chaperone and, eventually, can cause apoptosis, for instance, in PC12 cells. Before the onset of initial apoptotic events, however, a marked decrease in the activity of extracellular signal-regulated kinases ERK 1/2 and protein kinase B/Akt can be observed together with reduced expression of the high affinity nerve growth factor receptor, tropomyosine-related kinase, TrkA, in this cell type. The proteasome inhibitor MG-132 can effectively counteract the geldanamycin-induced reduction of TrkA expression and it can render TrkA and ERK1/2 phosphorylation but not that of protein kinase B/Akt by nerve growth factor again inducible. We have found altered intracellular distribution of TrkA in geldanamycin-treated and proteasome-inhibited PC12 cells that may, at least from the viewpoint of protein localization explain why nerve growth factor remains without effect on protein kinase B/Akt. The lack of protein kinase B/Akt stimulation by nerve growth factor in turn reveals why nerve growth factor treatment cannot save PC12 cells from geldanamycin-induced programmed cell death. Our observations can help to better understand the mechanism of action of geldanamycin, a compound with strong human therapeutical potential.

A simple fluorescent labeling technique to study virus adsorption in Newcastle disease virus infected cells.

The present study demonstrates that the fluorescent general membrane dyes PKH67 and PKH26 are suitable to label Newcastle disease virus, an enveloped virus belonging to the family of paramyxoviridae. Adsorption of the labeled virus particles was tracked, visualized and quantitated using confocal laser scanning microscopy. The specificity of PKH-labeling was determined by colocalization analysis of the PKH signal with NDV-specific immunolabeling, and by using mock-infected controls and infection with detergent-pretreated labeled virus particles. The infectivity of the NDV particles was not affected by the labeling procedure as indicated by the results of a cytotoxicity ATP assay, an apoptosis assay and detection of virus-specific RNA and protein by qPCR and Western blotting, respectively, in cells infected with PKH-labeled and unlabeled virus particles. This technique can be used as an inexpensive, sensitive and rapid alternative method in the analysis of adsorption and internalization of enveloped viruses by the infected cells.